The present invention relates to methods employing an ADP receptor, termed the P2Y12 receptor, and its encoding nucleic acid molecules. The invention also relates to methods for the recombinant production of the receptor proteins and the proteins made by these methods, antibodies against the whole receptor or regions thereof, vectors, nucleotide probes, host cells transformed by genes encoding polypeptides having the receptor activity, methods for the discovery of novel modulators of receptor activity, along with diagnostic and therapeutic uses for these various agents.
Thrombotic complications are a major cause of death in the industrialized world. Examples of these complications include acute myocardial infarction, unstable angina, chronic stable angina, transient ischemic attacks, strokes, peripheral vascular disease, preeclampsia, deep venous thrombosis, embolism, disseminated intravascular coagulation and thrombotic thrombocytopenic purpura. Thrombotic and restenotic complications also occur following invasive procedures, e.g., angioplasty, carotid endarterectomy, post CABG (coronary artery bypass graft) surgery, vascular graft surgery, stent placements and insertion of endovascular devices and prostheses.
It is generally thought that platelet aggregates play a critical role in these events. Blood platelets, which normally circulate freely in the vasculature, become activated and aggregate to form a thrombus with disturbed blood flow caused by ruptured atherosclerotic lesions or by invasive treatments such as angioplasty, resulting in vascular occlusion. Platelet activation can be initiated by a variety of agents, e.g., exposed subendothelial matrix molecules such as collagen, or by thrombin which is formed in the coagulation cascade.
An important mediator of platelet activation and aggregation is ADP (adenosine 5xe2x80x2-diphosphate) which is released from blood platelets in the vasculature upon activation by various agents, such as collagen and thrombin, and from damaged blood cells, endothelium or tissues. Activation of platelets by ADP results in the recruitment of more platelets and stabilization of existing platelet aggregates. Platelet ADP receptors mediating aggregation are activated by ADP and some of its derivatives and antagonized by ATP (adenosine 5xe2x80x2-triphosphate) and some of its derivatives (Mills, D. C. B. (1996) Thromb. Hemost. 76:835-56). Therefore, platelet ADP receptors are members of the family of P2 receptors activated by purine and/or pyrimidine nucleotides (King, B. F., Townsend-Nicholson, A. and Burnstock, G. (1998) Trends Pharmacol. Sci. 19:506-514). ADP receptors additionally have been characterized as belonging to the G protein-coupled receptor superfamily.
Studies of inherited disorders in humans and rats which result in a reduction of ADP release from platelets or reduced ADP receptor number and signaling confirm the critical role in platelet aggregation of ADP and the ADP receptors (Cattaneo M. and Gachet C., Arterioscler. Thromb. Vasc. Biol. (1999) 19:2281-2285). Inhibitors of ADP-induced platelet aggregation are efficacious antithrombotic drugs.
Recent pharmacological data using selective antagonists suggests that ADP-dependent platelet aggregation requires activation of at least two ADP receptors (Kunapuli, S. P. (1998) TIPS 19:391-394; Kunapuli S. P. et al., (1998) Biochem J. 336:513-523; Jantzen, H. M. et al., (1999) Thromb. Haemost. 81:111-117). One receptor appears to be identical to the cloned P2Y1 receptor, is coupled to the G protein Gq and mediates intracellular calcium mobilization. This receptor is also required for ADP-induced platelet shape change.
The second platelet ADP receptor couples to inhibition of adenylyl cyclase. The gene or cDNAs for this receptor has not been identified previously and the receptor has been provisionally termed P2YADP (Fredholm B. B. et al., (1997) TIPS 18:79-82), P2TAC, (Kunapuli, S (1998) TIPS 19(10):391-394), and P2Ycyc based on its pharmacological and signaling properties. Based on the inhibition of adenylyl cyclase and the activation of the G protein Gxcex1i2 by ADP in platelet membranes it was thought that this receptor may couple to Gi in vivo, and may belong to the G-protein coupled receptor family. This receptor also appears to be the target of the orally active antithrombotic drugs ticlopidine and clopidogrel, which appear to act through unstable and irreversible acting liver metabolites (Quinn, M. J. and Fitzgerald, D. J. (1999) Circulation 100:1667-1672). Patients with mild bleeding disorders have been identified with functional defects in this receptor (Cattaneo M. and Gachet C., Arterioscler. Thromb. Vasc. Biol. (1999) 19:2281-2285). The molecular identity of the Gi-linked receptor has remained elusive, even though it is the target of efficacious antithrombotic agents such as ticlopidine and clopidogrel (Gachet et al. (1990)). A sequence corresponding to this Gi-linked receptor was previously published in WO 98/50549, but its function was not identified.
Despite the previous physiological identification of a second ADP receptor and the discovery of agents that may act at this receptor, there exists a need for platelet ADP receptor inhibitors with improved properties.
The present inventors have cloned the Gi-linked receptor, designated P2Y12, and show that a patient with a bleeding disorder (Nurden et al (1995)) is defective in this gene. Cloning of the P2Y12 receptor facilitates the development of better antiplatelet agents to treat a variety of cardiovascular diseases.
The invention includes an isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2, an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 12 (b) an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NO: 1 under conditions of sufficient stringency to produce a clear signal; and (c) an isolated nucleic acid molecule which hybridizes to a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2 under conditions of sufficient stringency to produce a clear signal.
The present invention further includes isolated nucleic acid molecules wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, isolated nucleic acid molecules that comprise nucleotides 130-1158 of SEQ ID NO: 1 or nucleotides 130-1161 of SEQ ID NO: 1; isolated nucleic acid molecules that comprise the sequence of SEQ ID NO: 11, nucleic acid molecules that comprise nucleotides 73-873 of SEQ ID NO: 11 or nucleotides 73-876 of SEQ ID NO: 11.
The present invention further includes the nucleic acids operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules. The invention further includes host cells transformed to contain the nucleic acids of the invention, wherein said host is selected selected from the group consisting of prokaryotic hosts and eukaryotic hosts and methods for producing a protein comprising the step of culturing a host cell under conditions in which the protein encoded by said nucleic acid molecule is expressed.
The invention further provides a polypeptide, which may be expressed in a recombinant cell or may be purified, selected from the group consisting of an isolated receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 2, an isolated receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 12.
The invention further provides an isolated antibody that binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies.
The invention further provides a method of identifying an agent which modulates the expression of a nucleic acid encoding a P2Y12 receptor comprising the steps of exposing cells which express the nucleic acid to the agent and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of P2Y12 receptor.
The invention further provides a method of identifying an agent which modulates at least one activity of a P2Y12 receptor comprising the steps of exposing cells which express the protein to the agent; determining whether the agent modulates at least one activity of said protein, thereby identifying an agent which modulates at least one activity of a P2Y12 receptor.
Activities the agent may modify include but are not limited to the interaction between ADP, ATP or a derivative thereof, and a P2Y12 receptor; potassium current or adenylyl cyclase activity, platelet activation or thrombotic activity; acute myocardial infarction, unstable angina, chronic stable angina, transient ischemic attacks, strokes, peripheral vascular disease, preeclampsia, deep venous thrombosis, embolism, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura or a bleeding disorder; thrombotic and restenotic complications following angioplasty, carotid endarterectomy, post CABG (coronary artery bypass graft) surgery, vascular graft surgery, stent placements or insertion of endovascular devices and prostheses.
The invention further provides a method of identifying binding partners for a P2Y12 receptor protein comprising the steps of exposing said protein to a potential binding partner and determining if the potential binding partner binds to said protein, thereby identifying binding partners for a P2Y12 receptor protein.
The invention further provides a method of modulating the expression of a nucleic acid encoding a P2Y12 receptor protein comprising the step of administering an effective amount of an agent which modulates the expression of a nucleic acid encoding encoding a P2Y12 receptor protein.
The invention further provides a method of modulating at least one activity of a P2Y12 receptor protein comprising the step of administering an effective amount of an agent which modulates at least one activity of a P2Y12 receptor protein.
The invention further provides a non-human transgenic animal modified to contain a nucleic acid molecule of the invention including a nucleic acid molecule which encode a P2Y12 receptor or a truncation mutant of P2Y12 receptor.
The invention further provides a method of diagnosing a disease state in a subject, comprising the step of determining the level of expression of a nucleic acid molecule encoding a P2Y12 receptor.
The invention further provides a method of diagnosing a disease state in a subject, comprising the step of determining the level of expression of a P2Y12 receptor protein.
The invention further provides an isolated mutant P2Y12 receptor protein which is a naturally occurring truncation mutant, wherein said truncation is caused by a frame-shift mutation in the region of the gene encoding transmembrane domain six of the protein.
The invention further provides an isolated mutant P2Y12 receptor protein which is a naturally occurring truncation mutant, wherein said truncation is caused by a frame-shift mutation in the region of the gene encoding transmembrane domain six of the protein wherein said protein is associated with a bleeding disorder.
The invention further provides an isolated mutant P2Y12 receptor protein which is a naturally occurring truncation mutant, wherein said truncation is caused by a frame-shift mutation in the region of the gene encoding transmembrane domain six of the protein wherein said frame shift mutation introduces a stop codon in the gene encoding the P2Y12 protein and results in platelets with impaired ADP-dependent platelet aggregation activity, reduced ADP binding activity and reduced ability to inhibit cAMP levels in response to ADP.
The invention further provides an isolated nucleic acid encoding a mutant P2Y12 receptor protein which is a naturally occurring truncation mutant, wherein said truncation is caused by a frame-shift mutation in the region of the gene encoding transmembrane domain six of the protein.
The invention further provides an isolated nucleic acid encoding a mutant P2Y12 receptor protein which is a naturally occurring truncation mutant, wherein said truncation is caused by a frame-shift mutation in the region of the gene encoding transmembrane domain six of the protein wherein said nucleic acid encodes a protein associated with a bleeding disorder.
The invention further provides an isolated nucleic acid encoding a mutant P2Y12 receptor protein which is a naturally occurring truncation mutant, wherein said truncation is caused by a frame-shift mutation in the region of the gene encoding transmembrane domain six of the protein wherein said frame shift mutation introduces a stop codon in the gene encoding the P2Y12 protein and results in platelets with impaired ADP-dependent platelet aggregation activity, reduced ADP binding activity and reduced ability to inhibit cAMP levels in response to ADP.
The invention further provides a method of diagnosing a disease state in a subject, comprising the step of determining the level of expression of a nucleic acid molecule of the invention.
The invention further provides a method of diagnosing a disease state in a subject, comprising the step of determining the level of expression of a protein of the invention.
The invention further provides a method of diagnosing a disease state in a subject, comprising the step of identifying a nucleic acid molecule of the invention.